Bacterial surface proteins have been used as carriers for foreign (heterologous) polypeptides (particularly in Salmonella and E. coli) for various purposes, including the development of live vaccines. In some instances, the heterologous material is expressed as a fusion product with a surface protein of the bacterium. Generally, the use of such surface proteins as a vehicle for expression and/or presentation of heterologous polypeptides has been limited by the characteristics of a particular surface protein. The lipopolysaccharide layer of a bacterium, which tends to stimulate a strong immune response, covers the integral outer membrane proteins of the organism and potentially affects efficient presentation of a cloned epitope. Where the surface protein is functional (for example, as part of a filamentous portion of a bacterial cell surface) there will be limited opportunities to express a fusion product and still retain the surface protein's function. Generally, the organisms that have been used for these purposes have been chosen because of the advantages presented in respect of the organism's relationship to its host.
Many genera of bacteria assemble layers composed of repetitive, regularly aligned, proteinaceous sub-units on the outer surface of the cell. These layers are essentially two-dimensional paracrystalline arrays, and being the outer molecular layer of the organism, directly interface with the environment. Such layers are commonly known as S-layers and are found on members of every taxonomic group of walled bacteria including: Archaebacteria; Chlamydia; Cyanobacteria; Acinetobacter; Bacillus; Acuaspirillum; Caulobacter; Clostridium; Chromatium. Typically, an S-layer will be composed of an intricate, geometric array of at least one major protein having a repetitive regular structure. In many cases, such as in Caulobacter, the S-layer protein is synthesized by the cell in large quantities and the S-layer completely envelopes the cell and thus appears to be a protective layer.
Caulobacter are natural inhabitants of most soil and freshwater environments and may persist in waste water treatment systems and effluents. The bacteria alternate between a stalked cell that is attached to a surface, and an adhesive motile dispersal cell that searches to find a new surface upon which to stick and convert to a stalked cell. The bacteria attach tenaciously to nearly all surfaces and do so without producing the extracelluar enzymes or polysaccharide "slimes" that are characteristic of most other surface attached bacteria. They have simple requirements for growth. The organism is ubiquitous in the environment and has been isolated from oligotrophic to mesotrophic situations. Caulobacters are known for their ability to tolerate low nutrient level stresses, for example, low phosphate levels. This nutrient can be limiting in many leachate waste streams, especially those with high levels of iron or calcium.
All of the freshwater Caulobacter that produce an S-layer are similar and have S-layers that are substantially the same. Such S-layers appear similar by electron microscopy with the layer being hexagonally arranged in all cases with a similar centre--centre dimension (see: Walker, S. G., et al. (1992). "Isolation and Comparison of the Paracrystalline Surface Layer Proteins of Freshwater Caulobacters" J. Bacteriol. 174: 1783-1792). 16S rRNA sequence analysis of several S-layer producing Caulobacter strains suggest that they group closely (see: Stahl, D. A. et al (1992) "The Phylogeny of Marine and Freshwater Caulobacters Reflects Their Habitat" J. Bacteriol. 174:2193-2198). DNA probing of Southern blots using the S-layer gene from C. crescentus CB15 identifies a single band that is consistent with the presence of a cognate gene (see: MacRae, J. D. and, J. Smit. (1991) "Characterization of Caulobacters Isolated from Wastewater Treatment Systems" Applied and Environmental Microbiology 57:751-758). Furthermore, antisera raised against the S-layer protein of C. crescentus strain CB15 reacts with S-layer proteins from other Caulobacter (see: Walker, S. G. et al (1992) [supra]). All S-layer proteins isolated from Caulobacter may be substantially purified using the same extraction method (pH extraction) which would not be expected to be a general purpose method for other bacterial membrane or surface associated proteins. All strains appear to have a polysaccharide reactive with antisera reactive against CB15 lipopolysaccharide species which appears to be required for S-layer attachment (see: Walker, S. G. et al (1992) [supra]).
The S-layer elaborated by freshwater isolates of Caulobacter are visibly indistinguishable from the S-layer produced by Caulobacter crescentus strains CB2 and CB15.
The S-layer proteins from the latter strains have approximately 100,000 m.w. although sizes of S-layer proteins from other species and strains will vary. The protein has been characterized both structurally and chemically. It is composed of ring-like structures spaced at 22nm intervals arranged in a hexagonal manner on the outer membrane. The S-layer is bound to the bacterial surface and may be removed by low pH treatment or by treatment with a calcium chelator such as EDTA.
The similarity of S-layer proteins in different strains of Caulobacter permits the use of a cloned S-layer protein gene of one Caulobacter strain for retrieval of the corresponding gene in other Caulobacter strains (see: Walker, S. G. et al (1992) [supra]; and, MacRae, J. D. et al (1991) [supra].
Expression, secretion and optionally, presentation, of a heterologous polypeptide as a fusion product with the S-layer protein of Caulobacter provides advantages not previously seen in systems using organisms such as E. coli and Salmonella where fusion products of other kinds of surface proteins have been expressed. All known Caulobacter strains are believed to be harmless and are nearly ubiquitous in aquatic environments. In contrast, many Salmonella and E. coli strains are pathogens. Consequently, expression and secretion of a heterologous polypeptide using Caulobacter as a vehicle will have the advantage that the expression system will be stable in a variety of outdoor environments and may not present problems associated with the use of a pathogenic organism. Furthermore, Caulobacter are natural biofilm forming species and may be adapted for use in fixed biofilm bioreactors. The quantity of S-layer protein that is synthesized and is secreted by Caulobacter is high, reaching 12% of the cell protein. The unique characteristics of the repetitive, two-dimensional S-layer would also make such bacteria ideal for use as an expression system, or as a presentation surface for heterologous polypeptides. This is desirable in a live vaccine to maximize presentation of the antigen or antigenic epitope. In addition, use of such a presentation surface to achieve maximal exposure of a desired polypeptide to the environment results in such bacteria being particularly suited for use in bioreactors or as carriers for the polypeptide in aqueous or terrestrial outdoor environments.